Clinical Chemistry
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 54: 1528-1536, 2008. First published July 24, 2008; 10.1373/clinchem.2008.104653
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
clinchem.2008.104653v1
54/9/1528    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chan, K. C. A.
Right arrow Articles by Lo, Y. M. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chan, K. C. A.
Right arrow Articles by Lo, Y. M. D.
(Clinical Chemistry. 2008;54:1528-1536.)
© 2008 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Quantitative Analysis of Circulating Methylated DNA as a Biomarker for Hepatocellular Carcinoma

K. C. Allen Chan1,2, Paul B. S. Lai3, Tony S. K. Mok1,4, Henry L. Y. Chan1,5, Chunming Ding6,7, S. W. Yeung2 and Y. M. Dennis Lo1,2,7,a

1 State Key Laboratory in Oncology in South China, Sir Y. K. Pao Centre for Cancer, Departments of 2 Chemical Pathology, 3 Surgery, 4 Clinical Oncology, and 5 Medicine and Therapeutics, 6 Stanley Ho Centre for the Emerging Infectious Diseases, and 7 Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong Special Administrative Region, China.

aAddress correspondence to this author at: Department of Chemical Pathology, Rm. 38023, 1/F, Clinical Science Building, Prince of Wales Hospital, 30-32 Ngan Shing St., Shatin, New Territories, Hong Kong SAR. Fax +852 2636 5090; e-mail loym{at}cuhk.edu.hk.


   Abstract
Top
 Abstract
Introduction
 Study Participants and Methods
 Results
 Discussion
 References
 
Background: Hypermethylation of the RASSF1A [Ras association (RalGDS/AF-6) domain family member 1A] gene is frequently observed in hepatocellular carcinoma (HCC). We evaluated the analysis of circulating hypermethylated RASSF1A for detecting HCC and assessing its prognosis.

Methods: In module 1, we studied 63 pairs of HCC patients and age- and sex-matched chronic hepatitis B virus (HBV) carriers, as well as 50 healthy volunteers. In module 2, we studied 22 HCC patients with cancer detected through a surveillance program. The concentrations of circulating hypermethylated RASSF1A sequences were measured by real-time PCR after digestion with a methylation-sensitive restriction enzyme.

Results: We detected hypermethylated RASSF1A sequences in the sera of 93% of HCC patients, 58% of HBV carriers, and 8% of the healthy volunteers. The median RASSF1A concentrations for the HCC patients and HBV carriers were 7.70 x 105 copies/L and 1.18 x 105 copies/L, respectively (P < 0.01). The detection of low concentrations in HBV carriers is consistent with previous findings that RASSF1A hypermethylation is an early event in HCC pathogenesis and can be found in premalignant liver tissues. Use of a marker cutoff value of 1 x 106 copies/L also identifies 50% of {alpha}-fetoprotein-negative HCC cases. Patients with higher RASSF1A concentrations at diagnosis or 1 year after tumor resection showed poorer disease-free survival (P < 0.01). For the HBV carriers who underwent HCC surveillance and subsequently developed HCC, the circulating concentration of RASSF1A increased significantly from the time of enrollment to cancer diagnosis (P = 0.014).

Conclusions: Detection and quantification of circulating methylated RASSF1A sequences are useful for HCC screening, detection, and prognostication.


   Introduction
Top
 Abstract
 Introduction
Study Participants and Methods
 Results
 Discussion
 References
 
The incidence of hepatocellular carcinoma (HCC),1 one of the commonest cancers worldwide (1), is increasing in the West (2). In China, where chronic infection with hepatitis B virus (HBV) is prevalent, the incidence of HCC is >30 cases per 100 000 population (3)(4). Despite numerous advances in the treatment of HCC during the last decade, the 5-year survival rate remains <40% (5)(6), and late presentation remains an important obstacle to successful treatment. In fact, many HCC patients have already developed locally advanced disease or distant metastasis by the time of presentation (5)(6). In this regard, biomarkers have been developed for early HCC detection (7), with {alpha}-fetoprotein (AFP) being the most widely used clinically. The diagnostic sensitivity of AFP for HCC is relatively low, however, and >40% of HCC patients have typical AFP concentrations at presentation (8). Therefore, the development of new biomarkers for early HCC detection is an important area of HCC research and has the potential to improve overall-survival rates.

Hypermethylation of tumor suppressor genes is frequently observed in HCC (9)(10). Such epigenetic changes are potential markers for detecting and monitoring HCC. Recently, we developed a method for the detection and quantification of circulating hypermethylated DNA sequences (11). This method combines the use of methylation-sensitive restriction enzyme digestion and real-time PCR detection. The use of this method with the tumor suppressor gene RASSF1A2 [Ras association (RalGDS/AF-6) domain family member 1A] as a model has demonstrated good diagnostic sensitivity and specificity for detecting placentally derived hypermethylated RASSF1A sequences in the plasma of pregnant women for the purpose of noninvasive prenatal diagnosis (11). In this study, we evaluated the clinical usefulness of this test for HCC detection and prognosis.

In the first module, we compared the serum concentrations of methylated RASSF1A sequences in HCC patients and 2 control groups (sex- and age-matched chronic HBV carriers and healthy volunteers) to evaluate the diagnostic sensitivity and specificity of this test. We also evaluated the prognostic value of methylated RASSF1A concentrations in the serum by conducting assays at several times after tumor resection. In the second module, we investigated the potential application of this test for HCC screening by measuring methylated RASSF1A concentrations in serum samples from 22 chronic HBV carriers who had undergone surveillance for HCC and had subsequently developed HCC.


   Study Participants and Methods
Top
 Abstract
 Introduction
 Study Participants and Methods
Results
 Discussion
 References
 
study participants
Module 1.
Sixty-three HCC patients undergoing surgical resection of HCC were recruited from the Department of Surgery, Prince of Wales Hospital. Three blood samples were collected from each HCC patient, at the time of diagnosis and at 1 month and 1 year after the surgery. The ethics approval committee of the institution approved the study. As a control, we recruited 1 chronic HBV carrier of the same sex and age for each HCC patient. We followed up all control individuals for at least 18 months, and none showed evidence of HCC. Table 1 summarizes the demographic information of the study participants. The high HBV prevalence in the HCC group is consistent with the fact that HBV infection is the most important etiologic factor for HCC in China (12). In contrast, the incidence of hepatitis C infection is very low. Fifty healthy individuals with no history of chronic hepatitis (median age, 48 years; 56% male) were recruited as another control group.


View this table:
[in this window]
[in a new window]

  		Table 1. Demographics of the HCC patients and the sex- and age-matched HBV carriers (N = 63).

Module 2.
A surveillance program for screening HCC in HBV carriers has been carried out at the Prince of Wales Hospital, Hong Kong. One thousand eighteen HBV carriers attending the hepatology clinic between 1997 and 2000 were screened for HCC by AFP measurement and abdominal ultrasonography (13). Twenty-three carriers developed HCC during this surveillance period, and serum samples were available from 22 of them. As controls, we recruited 22 sex- and age-matched individuals who had enrolled in the surveillance program but who did not develop HCC. The median age of both groups was 56 years (interquartile range, 45–62 years), and 19 (86%) of these controls were male. The details of the surveillance program have been described elsewhere (13).

sample processing
HCC tissues and blood cells.
To verify the specificity of the methylation-sensitive restriction enzyme-mediated real-time PCR system that we used to detect methylated DNA sequences, we used this detection system and bisulfite sequencing to analyze the methylation status of the RASSF1A and ACTB (actin, beta) promoters in tumor tissues and blood cells from 5 HCC patients.

Serum samples.
DNA was extracted from 800 µL of serum with the QIAamp DNA Mini Kit (Qiagen) as previously described (14)(15).

digestion with a methylation-sensitive restriction enzyme
We digested 100 ng of DNA from HCC tissues and blood cells or 35 µL of extracted serum DNA with 100 U of BstUI, a methylation-sensitive restriction enzyme, at 60 °C for 16 h. The methylation-sensitive enzyme degrades unmethylated DNA sequences, whereas methylated DNA sequences remain intact and detectable by real-time PCR.

real-time detection and quantification of rassf1a and actb sequences
We used a duplex real-time PCR assay to detect RASSF1A and ACTB sequences simultaneously (11). The sequences of the forward and reverse primers and the probe for RASSF1A are 5'-AGCCTGAGCT CATTGAGCTG-3', 5'-ACCAGCTGCCGTGTGG-3', and 5'FAMTM-CCAACGCGCTGCGCAT(MGBTM)-3', respectively. The corresponding primer and probe sequences for ACTB are 5'-GCGCCGTTCCGAAAGTT-3', 5'-CGGCGGATCGGCAAA-3', and 5'VICTM-ACCG CCGAGACCGCGTC(MGBTM)-3', respectively. FAM and VIC are proprietary fluorescent dyes (Applied Biosystems), and MGB is a proprietary quencher (Applied Biosystems). Each reaction contained 1x TaqMan Universal PCR Master Mix (Applied Biosystems), 300 nmol/L of each RASSF1A primer, 85 nmol/L of the RASSF1A probe, 450 nmol/L of each ACTB primer, and 126 nmol/L of the ACTB probe. We used 5 µL of DNA not treated with enzyme or 7.15 µL of an enzyme-digested serum DNA mixture (equivalent to 5 µL of undigested serum DNA) as the template for each reaction. The thermal profile was 50 °C for 2 min, 95 °C for 10 min, and 50 cycles of 95 °C for 15 s and 60 °C for 1 min (11). We ran all reactions in duplicate and calculated the mean quantity. A DNA construct containing 1 copy each of the RASSF1A and ACTB amplicons was established as the quantitative calibrator.

bisulfite sequencing of the rassf1a and actb promoters in tumor tissues and blood cells
We treated 1 µg of DNA with bisulfite, amplified the RASSF1A and ACTB promoters, and then cloned and sequenced the PCR products. CpG sites with cytosine residues sequenced as cytosine or thymine were scored as methylated or unmethylated, respectively.

precision of the analysis of serum methylated rassf1a
To investigate the reproducibility of this test for quantifying the circulating concentration of methylated RASSF1A, we evaluated the CV for the methylated RASSF1A assay. We digested 20 aliquots of a DNA sample extracted from an HCC tumor tissue with BstUI enzyme and quantified the methylated RASSF1A concentration by real-time PCR. The CV of the 20 replicate analyses was 11%.

statistical analysis
We compared the serum RASSF1A concentrations of HCC patients and matched HBV carriers with the Wilcoxon signed rank test and multiple logistic regression analysis, and we used the log-rank test and Cox proportional hazards regression analysis for survival analyses. Classification and regression tree analysis was used to evaluate RASSF1A cutoff values for combining the use of the AFP marker and RASSF1A analysis. P values <0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Study Participants and Methods
 Results
Discussion
 References
 
concordant results for enzyme-mediated real-time analysis and bisulfite sequencing
To evaluate the specificity of this new system for detecting hypermethylated DNA sequences, we analyzed tumor tissues and blood cells from 5 HCC patients with bisulfite sequencing and with the enzyme-mediated real-time detection system. In the bisulfite-sequencing analysis, we detected RASSF1A promoter hypermethylation in all tumor tissues but in none of the blood cell samples. In contrast, the ACTB promoter was completely unmethylated in both sample types. The results of the enzyme-digestion analysis were in complete accord with the bisulfite-sequencing results. After enzyme digestion, RASSF1A sequences were detectable only in the tumor tissues, whereas ACTB sequences were detectable in neither tumor tissues nor blood cells. Fig. 1 shows the results for bisulfite sequencing and real-time PCR analysis for 1 case.


Figure 1
View larger version (61K):
[in this window]
[in a new window]

  		Figure 1. Bisulfite-sequencing (left) and the corresponding real-time amplification analysis (right) results of a representative HCC patient.

For the bisulfite-sequencing analysis, each row represents 1 DNA molecule. The CpG sites are numbered according to their sequence in the PCR amplicon of the particular assay. The first CG dinucleotide in the PCR amplicon is numbered as 1. The CpG sites within recognition sites of the methylation-sensitive restriction enzyme are underlined. Open and closed circles represent unmethylated and methylated CpG sites, respectively.

detection of serum actb sequences after enzyme digestion
We incorporated a system in our analysis to check the completeness of enzyme digestion to minimize the chance of false-positive results caused by incomplete digestion of the background unmethylated RASSF1A sequences. This checking system consisted of a PCR assay that amplified the ACTB promoter with the same number of restriction sites as in the RASSF1A amplicon. Because we have previously shown that the digestion efficiencies for RASSF1A and ACTB sequences are similar (11), the presence of an ACTB signal in a sample suggests that the digestion of unmethylated RASSF1A is incomplete. In such cases, the DNA samples would be redigested for 8 h. Serum RASSF1A results would be used for further analysis only if the corresponding ACTB signal for the sample was absent. Of the 302 samples analyzed in module 1, 32 samples (11%) showed a detectable ACTB signal after the initial digestion. There were no significant differences in the rate of incomplete digestion between samples from HCC patients, HBV carriers, and healthy control individuals (P = 0.53, {chi}2 test). Of the 88 samples analyzed in module 2, 13 samples (15%) showed incomplete enzyme digestion in the initial analysis. No sample showed detectable ACTB signal after redigestion. To investigate whether redigestion of a DNA sample appreciably reduces the amount of methylated RASSF1A, we digested 20 samples of DNA extracted from HCC tissues with BstUI for 16 h. For each sample, we used half of the DNA mixture for real-time PCR and further digested the other half for 8 h before quantifying RASSF1A. The RASSF1A concentrations obtained for single and repeat digestions of samples were not significantly different (P = 0.106, paired Student t-test).

detection of rassf1a sequence after digestion of serum samples from hcc patients
In module 1, RASSF1A sequences were detectable after enzyme digestion in the sera of 93% (59 of 63) of the HCC patients at diagnosis and in 58% (37 of 63) of the HBV carriers. The median concentrations for the 2 groups were 7.70 x 105 copies/L and 1.18 x 105 copies/L, respectively (P < 0.0001, Wilcoxon test). The difference between the 2 groups in the total serum concentration of RASSF1A (without enzyme digestion) was not statistically significant (P = 0.5, Wilcoxon test). Only 4 (8%) of the 50 healthy individuals showed detectable RASSF1A after digestion. The median concentration for these 4 individuals was 3.7 x 104 copies/L (Fig. 2A ). The area under the ROC curve for distinguishing between HCC patients and HBV carriers was 0.81.


Figure 2
View larger version (17K):
[in this window]
[in a new window]

  		Figure 2. Serum concentrations of RASSF1A sequences after digestion in HCC patients, HBV carriers, and healthy individuals (A) and in HCC patients and HBV carriers with and without ultrasonographic evidence of cirrhosis (B).

Brackets and P values refer to the indicated group comparisons. The upper, middle, and lower lines of each box represent the 75th, 50th, and 25th percentiles, respectively. The upper and lower whiskers represent the 95th and 5th percentiles.

effect of cirrhosis on the serum rassf1a concentration
HBV carriers with ultrasonographic evidence of cirrhosis had higher serum RASSF1A concentrations than those without cirrhosis (median, 1.32 x 105 copies/L vs 0 copies/L; P = 0.032, Mann-Whitney U-test; Fig. 1BUp ). In contrast, serum RASSF1A concentrations for HCC patients with and without ultrasonographic evidence of cirrhosis were not statistically different (median, 7.98 x 105 copies/L vs 7.00 x 105 copies/L; P = 0.529, Mann-Whitney U-test; Fig. 2BUp ). The difference in serum RASSF1A concentrations between HCC patients and HBV carriers was independent of the presence of ultrasonographic and biochemical changes due to cirrhosis (logistic regression; see Table 1Up ).

serum rassf1a concentrations after surgical resection
Of the 59 patients with detectable methylated RASSF1A in the serum at diagnosis, 45 patients (76%) showed a reduced concentration 1 month after tumor resection. The median RASSF1A concentration decreased from 7.70 x 105 copies/L to 2.50 x 105 copies/L (P < 0.0001, Wilcoxon test).

disease-free survival
Patients with serum RASSF1A concentrations greater than the median concentration of the group at diagnosis showed significantly poorer disease-free survival (defined as survival without any clinical evidence of disease recurrence) than the patients with lower concentrations (P = 0.0034, log-rank test; Fig. 3A ). The difference in survival probabilities was independent of the presence of biochemical and ultrasonographic evidence of cirrhosis (P = 0.0028, Cox proportional hazards regression). Similarly, the RASSF1A concentration 1 year after surgery was also associated with survival (P = 0.031, log-rank test; Fig. 3C ). On the other hand, the concentration 1 month after the operation was not associated with survival (P = 0.33, log-rank test; Fig. 3B ).


Figure 3
View larger version (14K):
[in this window]
[in a new window]

  		Figure 3. Disease-free survival probabilities in HCC patients according to the serum RASSF1A concentration after enzyme digestion at diagnosis (A) and at 1 month (B) and 1 year (C) after tumor resection.

Disease-free survival is defined as survival with no clinical evidence of disease recurrence. For each time point, the patients were divided into 2 groups with the median concentration for all patients as a cutoff value. The median concentrations at diagnosis and at 1 month and 1 year after the operation were 7.70 x 105, 2.50 x 105, and 9.10 x 105 copies/L, respectively. For the survival analysis with the serum concentration at 1 year, only the patients with no clinical evidence of residual disease at 1 year were analyzed.

correlation between the serum concentrations of afp and rassf1a
The serum concentrations of RASSF1A and AFP were not significantly correlated, either for the HCC patients (P = 0.5, Spearman correlation analysis) or for the chronic HBV carriers (P = 0.91, Spearman correlation analysis), suggesting that concurrent use of both markers might provide a nonoverlapping and potentially synergistic set of information. The cutoff values for methylated RASSF1A in serum were determined by classification and regression tree analysis for classifying individuals with typical or increased AFP concentrations. For individuals with increased AFP, we used a lower RASSF1A cutoff value of 1.50 x 105 copies/L to screen out false-positive AFP results, and we used a higher RASSF1A cutoff value of 1 x 106 copies/L for individuals with typical AFP concentrations to identify HCC patients with false-negative AFP results. Twenty-two (35%) of the 63 HCC patients in module 1 had typical AFP concentrations of <20 µg/L. A serum RASSF1A cutoff value of 1 x 106 copies/L further identified 11 patients (50%) who had false-negative AFP results (Fig. 4 ). On the other hand, 4 of the 8 HBV carriers with increased AFP values had RASSF1A concentrations of <1.50 x 105 copies/L.


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		Figure 4. Classification of 63 HCC patients and 63 sex- and age-matched chronic HBV carriers according to their postdigestion serum concentration of RASSF1A (copies per liter) and AFP (micrograms per liter).

Twenty-two HCC patients had typical AFP concentrations (<20 µg/L), and 19 patients (86%) had increased RASSF1A concentrations (>1.50 x 105 copies/L).

serum rassf1a concentrations in hbv carriers under surveillance for hcc
In module 2, we studied 22 HBV carriers who developed HCC during the HCC screening program. The median time between enrollment and HCC diagnosis was 30 months, and we observed a significant increase in serum RASSF1A concentration from enrollment to HCC diagnosis (median, 3.74 x 105 copies/L to 5.05 x 105 copies/L; P = 0.014, Wilcoxon test; Fig. 5 ). For the matched control individuals who had not developed HCC, the serum RASSF1A concentrations at the 2 corresponding time points were not significantly different (median, 1.16 x 105 copies/L vs 0.92 x 105 copies/L; P = 0.495). At the second time point, the patients who had developed HCC had significantly higher methylated RASSF1A concentrations in the serum than the control individuals (P < 0.001, Wilcoxon test).


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		Figure 5. Serum RASSF1A concentrations in chronic HBV carriers undergoing HCC surveillance who did and did not develop HCC.

For the group with HCC development, serum RASSF1A concentrations were measured at the time of enrollment and when HCC was confirmed. For the group without HCC development, the second measurement was taken as close as possible to the time of HCC diagnosis for the corresponding HCC patient.

Twelve (60%) of the 22 patients who had developed HCC were classified as having HCC with the combined AFP and RASSF1A algorithm (Fig. 4Up ) at the time of HCC confirmation. In comparison, AFP measurement alone identified only 8 cases (40%). Moreover, only 1 of the 22 control individuals fulfilled the combined criteria for HCC at the time of enrollment; none fulfilled these criteria at follow-up.


   Discussion
Top
 Abstract
 Introduction
 Study Participants and Methods
 Results
 Discussion
References
 
Hypermethylation of the RASSF1A promoter is frequently observed in HCC and has been shown to play an important role in HCC pathogenesis (9)(16)(17); however, methylation-specific PCR detects hypermethylated RASSF1A sequences in the circulation of only 40%–70% of HCC patients (18)(19). These low detection rates are probably related to the substantial degradation of DNA (up to 96%) caused by the bisulfite conversion step used in methylation-specific PCR (20) and the low concentration of circulating tumor DNA in cancer patients (21)(22). We recently developed a nonbisulfite method for detecting and quantifying circulating hypermethylated RASSF1A sequences (11). With this method, we detected methylated RASSF1A sequences in 93% of HCC patients. This frequency is similar to that previously reported for detecting RASSF1A hypermethylation in HCC tumor tissues (9)(18). The substantial improvement is likely to be related to specific degradation of unmethylated sequence by the methylation-sensitive restriction enzyme, in contrast to the nondiscriminatory degradation of both methylated and unmethylated DNA with bisulfite conversion (20). This improvement also has enhanced the reliability of the quantitative measurement. Thus, our study is the first to show that the concentration of circulating hypermethylated RASSF1A is associated with disease-free survival in HCC patients after tumor resection.

Despite the extensive use of methylation-sensitive restriction enzymes in epigenetic research, methylation-sensitive restriction enzyme analysis has not been popular for detecting aberrant DNA methylation in the circulation for the purpose of cancer detection. The reason is probably related to the difficulties in differentiating false-positive results due to incomplete enzyme digestion from the presence of a low concentration of tumor-derived hypermethylated sequences. In this regard, we incorporated several measures to minimize the potential problem of incomplete digestion. These measures included (a) the inclusion of multiple enzyme restriction sites in the PCR amplicon, (b) the use of excess restriction enzyme and a prolonged incubation period, and (c) the introduction of a system to check the completeness of enzyme digestion. Our check for incomplete digestion demonstrated >10% of the samples are potentially incompletely digested after the initial enzyme-digestion step.

The presence of low concentrations of methylated RASSF1A in the sera of 58% of the chronic HBV carriers is consistent with previous reports that RASSF1A promoter hypermethylation is an early event in HCC pathogenesis and can be observed in nonmalignant liver tissues of patients with cirrhosis or chronic hepatitis (16)(23). On the other hand, the predominant source of circulating methylated RASSF1A in HCC patients is likely the tumor cells for the following reasons. First, the median concentration of methylated RASSF1A in the serum was >6-fold higher in HCC patients than in matched HBV carriers, a difference that was independent of the presence of cirrhosis. Second, a significant reduction in serum RASSF1A was observed in the HCC patients after tumor resection. Lastly, we observed a significant increase in the circulating concentration of methylated RASSF1A from the time of recruitment to the time of HCC development in the HBV carriers who subsequently developed HCC.

Diagnostically, the absence of a correlation between the serum concentrations of AFP and RASSF1A has allowed the synergistic use of these 2 biomarkers to improve their diagnostic accuracies. The use of classification and regression tree analysis has produced a different serum RASSF1A cutoff values for individuals with increased AFP concentrations than for those without increased AFP concentrations. The diagnostic sensitivity and specificity were 77% and 89%, respectively, for this combined AFP and RASSF1A analysis, compared with 65% and 87%, respectively, for AFP measurement alone. This improved diagnostic accuracy may be useful for early detection of HCC, because increased AFP concentrations have been shown in only about 40% of HCC patients with early-stage disease (8).

In addition to HCC detection, analysis of methylated RASSF1A in the serum also has prognostic value in that the concentrations in these patients at diagnosis and 1 year after surgery are associated with disease-free survival. This prognostic value was also independent of the presence of ultrasonographic and biochemical evidence of cirrhosis. These findings suggest that methylated RASSF1A in the serum reflects the tumor load in HCC patients. A correlation between circulating tumor DNA and tumor load has also been described for other cancers (24)(25). The apparent lack of a correlation between the serum concentration of RASSF1A at 1 month after the operation and survival may be related to the background concentration of methylated RASSF1A derived from nonmalignant hepatitic liver tissues. In the presence of a tumor, a high proportion of the circulating RASSF1A would be derived from tumor cells, and hence the RASSF1A concentration could reflect the tumor load. After tumor resection, however, the methylated RASSF1A sequences released from nonmalignant hepatitic liver tissue may obscure the small amounts of RASSF1A sequences derived from residual cancer cells. Because the RASSF1A concentration observed at 1 year after tumor resection (but not the concentration at 1 month) was associated with disease recurrence, monitoring the changes in RASSF1A may be useful for identifying disease recurrence.

In this study, we demonstrated the clinical usefulness of the methylation-sensitive restriction enzyme-mediated PCR approach for detecting aberrantly methylated tumor suppressor gene sequences in the circulation of cancer patients. This method permitted the detection of methylated RASSF1A sequences in the sera of 93% of HCC patients. This detection rate is better than that reported for bisulfite-based detection methods. Adopting different cutoff values for individuals with typical and increased AFP concentrations may facilitate the use of serum RASSF1A analysis for HCC detection. Although hypermethylation of the RASSF1A promoter is not specific for HCC (26)(27)(28)(29), in the context of HCC screening in a chronic hepatitis carrier, an observed increase in the circulating concentration of methylated RASSF1A would definitely warrant further investigation for HCC, as via an imaging study for example. The presence of very low concentrations of methylated RASSF1A in the healthy control individuals is an interesting finding. Previous studies have shown that hypermethylation of the RASSF1A promoter may be present in a very low percentage of premalignant lesions (30) and even nonpathologic tissues (31). Diagnostically, none of these healthy individuals had circulating concentrations of methylated RASSF1A greater than the cutoff of 1.50 x 105 copies/L proposed for HCC diagnosis. Technically, this method is relatively simple and inexpensive and hence has the potential to be adapted for routine clinical use. More importantly, the application of this technology would not be limited to detecting aberrantly methylated RASSF1A for HCC detection only. Because aberrant methylation of tumor suppressor genes has been extensively reported for virtually all cancers, this technology can be generalized for the detection of different types ofcancer via the use of different panels of tumor suppressor genes (26)(27)(28)(29).


   Acknowledgments
 
Grant/Funding Support: This work has been supported by the Innovation and Technology Commission of the Hong Kong SAR Government under the Innovation and Technology Support Program (ITS/014/06).

Financial Disclosures: The authors have filed a patent application on the technology described in this report.


   Footnotes
 
1 Nonstandard abbreviations: HCC, hepatocellular carcinoma; HBV, hepatitis B virus; AFP, {alpha}-fetoprotein.

2 Human genes: RASSF1A, Ras association (RalGDS/AF-6) domain family member 1A; ACTB, actin, beta.


   References
Top
 Abstract
 Introduction
 Study Participants and Methods
 Results
 Discussion
 References
 

  1. El-Serag HB. Hepatocellular carcinoma: an epidemiologic view. J Clin Gastroenterol 2002;35:S72-S78.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  2. El-Serag HB, Davila JA, Petersen NJ, McGlynn KA. The continuing increase in the incidence of hepatocellular carcinoma in the United States: an update. Ann Intern Med 2003;139:817-823.[Abstract/Free Full Text]
  3. Merican I, Guan R, Amarapuka D, Alexander MJ, Chutaputti A, Chien RN, et al. Chronic hepatitis B virus infection in Asian countries. J Gastroenterol Hepatol 2000;15:1356-1361.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  4. Beasley RP, Hwang LY, Lin CC, Chien CS. Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22 707 men in Taiwan. Lancet 1981;2:1129-1133.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  5. Kung JW, Macdougall M, Madhavan KK, Garden OJ, Parks RW. Predicting survival in patients with hepatocellular carcinoma: a UK perspective. Eur J Surg Oncol 2007;33:188-194.[CrossRef][Medline] [Order article via Infotrieve]
  6. Chen CH, Su WW, Yang SS, Chang TT, Cheng KS, Lin HH, et al. Long-term trends and geographic variations in the survival of patients with hepatocellular carcinoma: analysis of 11,312 patients in Taiwan. J Gastroenterol Hepatol 2006;21:1561-1566.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  7. Sherman M. Diagnosis of small hepatocellular carcinoma. Hepatology 2005;42:14-16.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  8. Farinati F, Marino D, De Giorgio M, Baldan A, Cantarini M, Cursaro C, et al. Diagnostic and prognostic role of alpha-fetoprotein in hepatocellular carcinoma: both or neither?. Am J Gastroenterol 2006;101:524-532.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  9. Schagdarsurengin U, Wilkens L, Steinemann D, Flemming P, Kreipe HH, Pfeifer GP, et al. Frequent epigenetic inactivation of the RASSF1A gene in hepatocellular carcinoma. Oncogene 2003;22:1866-1871.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  10. Yang B, Guo M, Herman JG, Clark DP. Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma. Am J Pathol 2003;163:1101-1107.[Abstract/Free Full Text]
  11. Chan KCA, Ding C, Gerovassili A, Yeung SW, Chiu RWK, Leung TN, et al. Hypermethylated RASSF1A in maternal plasma: a universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis. Clin Chem 2006;52:2211-2218.[Abstract/Free Full Text]
  12. Gibson JB, Wu PC, Ho JC, Lauder IJ. Hepatitis B surface antigen, hepatocellular carcinoma and cirrhosis in Hong Kong: a necropsy study: 1963–1976. Br J Cancer 1980;42:370-377.[Web of Science][Medline] [Order article via Infotrieve]
  13. Mok TS, Yeo W, Yu S, Lai P, Chan HL, Chan AT, et al. An intensive surveillance program detected a high incidence of hepatocellular carcinoma among hepatitis B virus carriers with abnormal alpha-fetoprotein levels or abdominal ultrasonography results. J Clin Oncol 2005;23:8041-8047.[Abstract/Free Full Text]
  14. Chan KCA, Zhang J, Chan ATC, Lei KIK, Leung SF, Chan LYS, et al. Molecular characterization of circulating EBV DNA in the plasma of nasopharyngeal carcinoma and lymphoma patients. Cancer Res 2003;63:2028-2032.[Abstract/Free Full Text]
  15. Chan KCA, Zhang J, Hui AB, Wong N, Lau TK, Leung TN, et al. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem 2004;50:88-92.[Abstract/Free Full Text]
  16. Lee S, Lee HJ, Kim JH, Lee HS, Jang JJ, Kang GH. Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. Am J Pathol 2003;163:1371-1378.[Abstract/Free Full Text]
  17. Yu J, Ni M, Xu J, Zhang H, Gao B, Gu J, et al. Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis. BMC Cancer 2002;2:29.[CrossRef][Medline] [Order article via Infotrieve]
  18. Yeo W, Wong N, Wong WL, Lai PB, Zhong S, Johnson PJ. High frequency of promoter hypermethylation of RASSF1A in tumor and plasma of patients with hepatocellular carcinoma. Liver Int 2005;25:266-272.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  19. Zhang YJ, Wu HC, Shen J, Ahsan H, Tsai WY, Yang HI, et al. Predicting hepatocellular carcinoma by detection of aberrant promoter methylation in serum DNA. Clin Cancer Res 2007;13:2378-2384.[Abstract/Free Full Text]
  20. Grunau C, Clark SJ, Rosenthal A. Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res 2001;29:E65.[CrossRef][Medline] [Order article via Infotrieve]
  21. Wong IH, Zhang J, Lai PBS, Lau WY, Lo YMD. Quantitative analysis of tumor-derived methylated p16INK4a sequences in plasma, serum, and blood cells of hepatocellular carcinoma patients. Clin Cancer Res 2003;9:1047-1052.[Abstract/Free Full Text]
  22. Chan KCA, Lo YMD. Circulating tumour-derived nucleic acids in cancer patients: potential applications as tumour markers. Br J Cancer 2007;96:681-685.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  23. Di Gioia S, Bianchi P, Destro A, Grizzi F, Malesci A, Laghi L, et al. Quantitative evaluation of RASSF1A methylation in the non-lesional, regenerative and neoplastic liver. BMC Cancer 2006;6:89.[CrossRef][Medline] [Order article via Infotrieve]
  24. Chan KCA, Chan ATC, Leung SF, Pang JCS, Wang AYM, Tong JH, et al. Investigation into the origin and tumoral mass correlation of plasma Epstein-Barr virus DNA in nasopharyngeal carcinoma. Clin Chem 2005;51:2192-2195.[Free Full Text]
  25. Lo YMD, Chan LYS, Lo KW, Leung SF, Zhang J, Chan ATC, et al. Quantitative analysis of cell-free Epstein-Barr virus DNA in plasma of patients with nasopharyngeal carcinoma. Cancer Res 1999;59:1188-1191.[Abstract/Free Full Text]
  26. Cho NY, Kim BH, Choi M, Yoo EJ, Moon KC, Cho YM, et al. Hypermethylation of CpG island loci and hypomethylation of LINE-1 and Alu repeats in prostate adenocarcinoma and their relationship to clinicopathological features. J Pathol 2007;211:269-277.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  27. Fischer JR, Ohnmacht U, Rieger N, Zemaitis M, Stoffregen C, Manegold C, Lahm H. Prognostic significance of RASSF1A promoter methylation on survival of non-small cell lung cancer patients treated with gemcitabine. Lung Cancer 2007;56:115-123.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  28. Taback B, Saha S, Hoon DS. Comparative analysis of mesenteric and peripheral blood circulating tumor DNA in colorectal cancer patients. Ann N Y Acad Sci 2006;1075:197-203.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  29. Wang Y, Yu Z, Wang T, Zhang J, Hong L, Chen L. Identification of epigenetic aberrant promoter methylation of RASSF1A in serum DNA and its clinicopathological significance in lung cancer. Lung Cancer 2007;56:289-294.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  30. Yeo W, Wong WL, Wong N, Law BK, Tse GM, Zhong S. High frequency of promoter hypermethylation of RASSF1A in tumorous and non-tumourous tissue of breast cancer. Pathology 2005;37:125-130.[Web of Science][Medline] [Order article via Infotrieve]
  31. Maruya S, Issa JP, Weber RS, Rosenthal DI, Haviland JC, Lotan R, El-Naggar AK. Differential methylation status of tumor-associated genes in head and neck squamous carcinoma: incidence and potential implications. Clin Cancer Res 2004;10:3825-3830.[Abstract/Free Full Text]



The following articles in journals at HighWire Press have cited this article:


Home page
 Neuro OncologyHome page
I. Lavon, M. Refael, B. Zelikovitch, E. Shalom, and T. Siegal
Serum DNA can define tumor-specific genetic and epigenetic markers in gliomas of various grades
Neuro Oncology, February 1, 2010; 12(2): 173 - 180.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
S. Huang, H. Li, X. Ding, and C. Xiong
Presence and Characterization of Cell-Free Seminal RNA in Healthy Individuals: Implications for Noninvasive Disease Diagnosis and Gene Expression Studies of the Male Reproductive System
Clin. Chem., November 1, 2009; 55(11): 1967 - 1976.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
T. deVos, R. Tetzner, F. Model, G. Weiss, M. Schuster, J. Distler, K. V. Steiger, R. Grutzmann, C. Pilarsky, J. K. Habermann, et al.
Circulating Methylated SEPT9 DNA in Plasma Is a Biomarker for Colorectal Cancer
Clin. Chem., July 1, 2009; 55(7): 1337 - 1346.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Pathol.Home page
E C W Hung, R W K Chiu, and Y M D Lo
Detection of circulating fetal nucleic acids: a review of methods and applications
J. Clin. Pathol., April 1, 2009; 62(4): 308 - 313.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
clinchem.2008.104653v1
54/9/1528    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chan, K. C. A.
Right arrow Articles by Lo, Y. M. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chan, K. C. A.
Right arrow Articles by Lo, Y. M. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS